This invention is generally directed to methods for the isolation of proteinase inhibitor proteins from plant tissues and, more particularly, to the isolation of heat stable proteinase inhibitor proteins from potato tubers.
Tubers from Solanaceous plants such as potato, are an abundant source of a wide range of classes of proteinase inhibitor proteins. Many of these proteins inhibit the activity of digestive proteinases, such as trypsin and chymotrypsin that naturally occur in both insects and mammals. By disturbing the natural digestive process, proteinase inhibitors form part of a plant""s natural defense against foraging by herbivores. For the same reason proteinase inhibitors have application in the pest control industry for the control of insects such as fire ants. In addition, because they also inhibit human digestive proteinases, these inhibitors have value in the pharmaceutical industry for the control of obesity and diabetes.
Potato tubers have been a major source for the study and preparation of proteinase inhibitors. Several of the proteinase inhibitors present in potato tubers are of the heat stable variety. Among these are two Kunitz-type proteinase inhibitors having a subunit Mr of about 20 and 20.5 kD that inhibit both trypsin and chymotrypsin (Walsh and Twichell, Plant Physiol. 97: 15-18, 1991). The smaller protein is a powerful inhibitor of chymotrypsin, and a weak inhibitor of trypsin. The larger protein is a powerful inhibitor of trypsin. Other heat stable proteinase inhibitors include one with a subunit Mr of about 9.5 kD (designated proteinase inhibitor I) which is a strong inhibitor of chymotrypsin (Melville and Ryan, J. Biol. Chem. 247:3445-3453, 1972), and another with a subunit Mr of about 10.5 kD (designated proteinase inhibitor II) which is a strong inhibitor of both chymotrypsin and trypsin (Bryant, Green and Ryan,. Biochemistry 15: 3418-3424, 1976).
Previous methods for preparation of these heat stable proteinase inhibitors have incorporated numerous steps, including: extraction in the presence of dithionate, heat, ammonium sulfate precipitation and chromatography (Walsh and Twichell, Plant Physiol. 97: 15-18, 1991; Melville and Ryan, J. Biol. Chem. 247: 3445-3453, 1972; Bryant, Green and Ryan, Biochemistry 15: 3418-3424, 1976; Ryan and Kassell, Methods in Enz. XIX: 883-889, 1970; Pearce and Ryan, Anal. Biochem. 130: 223-225, 1983). Unfortunately these methods are cumbersome, tedious, time consuming, expensive and produce relatively low yields of heat stable proteinase inhibitors. A major problem is that initial extracts form a pasty homogenate with poor flow characteristics resulting in difficulties with subsequent processing steps, especially filtration, ammonium sulfate precipitation and resolubilization. The pasty consistency sometimes requires the use of pressurized filtration during subsequent steps, especially in larger scale extractions (Ryan and Kassell, 1970). Furthermore, the pasty consistency reduces yield because of difficulty in fully recovering material from the paste. These previous methods are also disadvantageous because they require skilled labor to execute and often take several days to complete.
Accordingly, there is a need in the art for a method of isolating proteinase inhibitors from plant tissue, especially potato tubers, that overcomes the problem of the pasty extract, eliminates the need for ammonium sulfate precipitation, is rapid, inexpensive, simple to perform and easy to accomplish on any scale from the laboratory to a large industrial process. The present invention fulfills these needs and provides other related advantages.
In brief, this invention relates to rapid and simple methods of isolating heat stable proteinase inhibitor proteins from plant tissues containing the same, particularly potato tubers. The method comprises three steps. First, proteins from potato tubers are extracted in soluble form in an aqueous/alcohol extraction medium such as dilute formic acid and 20% ethanol. The presence of the ethanol in the extraction medium converts the otherwise pasty homogenate to a smooth flowing alcohol extract that is easy to manipulate and particularly easy to filter. Second, the alcohol is heated to a first temperature then cooled to a second temperature. Heating in the presence of alcohol denatures most of the unwanted proteins in the alcohol extract, and subsequent cooling leads to formation of a precipitate phase constituting debris and a soluble phase that contains the heat stable proteinase inhibitor proteins. Third, the heat stable proteinase inhibitor proteins are precipitated from the soluble phase by dialysis against a suitable dialysis medium, such as dilute formic acid or water followed by dilute formic acid. The precipitated proteins may be either a single inhibitor proteinxe2x80x94proteinase inhibitor II, or a mixture of proteinase inhibitor II and two Kunitz proteinase inhibitors (one most active against trypsin and the other most active against chymotrypsin). The precipitated proteinase inhibitor proteins are free of the bulk of other proteins and other constituents originally present in the tuber.
Whether the precipitated proteins constitute the single inhibitor or the mixture of inhibitors is determined by a single modification. This modification relates to the heating temperature selected for the denaturation step. The single inhibitor may be obtained upon heating to 70xc2x0 C., cooling to 50xc2x0 C. followed by dialysis against 0.22% formic acid. The inhibitor mixture may be obtained by heating to 50xc2x0 C., cooling to room temperature followed by dialysis against water, where the water dialysis is further followed by the addition formic acid to 0.88%.
Excluding dialysis time, the entire method can be performed in less than an hour compared with the days that were required for previous methods. The method is simple, can be performed by unskilled technicians and is amenable to performance on any scale. It avoids the use of expensive ammonium sulfate and chromatography steps, and even the ethanol can be recovered which further reduces the process costs of the method. The method can yield 300 mg of substantially pure proteinase inhibitor proteins from 1 pound of potatoes which is vastly superior to the 150 mg yield of less pure proteins from 100 pounds of potatoes using prior methods.